1. Field of the Invention
The present invention relates to methods and apparatus for cell activity assay (CAA) investigation of chemotaxis, migration, invasion, angiogenesis, growth, proliferation, differentiation, or interaction of cells in response to various chemical environments.
2. Background of the Invention
Chemotaxis is the directional movement (migration) of biological cells or organisms in response to concentration gradients of chemicals. Invasion is the movement (migration) of cells into or through a barrier. Tumor invasion is such action initiated by cancer cells into or through biological tissue in vivo, or, into or through extra cellular matrix proteins, e.g., collagen or matrigel, into or through barriers made of other substances, in vitro. Angiogenesis is the migration and formation of capillary blood vessels by endothelial cells. Growth is the increase in the size, form, or complexity of cells. Proliferation is growth of cells by cell division. Differentiation is the process by which cells change from a less specialized to a more specialized state usually associated with different functional roles and the expression of new and different traits. Interaction of cells is the alteration of cell behavior such as movement, invasion, angiogenesis, growth, proliferation, or differentiation in response to the presence and action of nearby cells of the same or different type. These activities and similar activities are referred to herein collectively as xe2x80x9ccell activity,xe2x80x9d and the apparatus employed to do the assays is referred to herein as xe2x80x9ccell activity assay apparatus.xe2x80x9d
One kind of single-site conventional cell activity assay apparatus referred to variously in the literature as xe2x80x9cchemotaxis chambers,xe2x80x9d xe2x80x9cBoyden chambers,xe2x80x9d xe2x80x9cBoyden chemotaxis chambers,xe2x80x9d xe2x80x9cblind well chambers.xe2x80x9d or xe2x80x9cmicrochemotaxis chambers,xe2x80x9d comprises two compartments separated by a membrane, with one or both of the compartments open to air. Multi-site apparatus are referred to as xe2x80x9cmulti-well chemotaxis chambers,xe2x80x9d or xe2x80x9cmulti-well Boyden chambers,xe2x80x9d and have the same basic site structure but have multiple sites. (See U.S. Pat. Nos. 5,210,021 and 5,302,515) Assays employing this kind of apparatus pipette cells suspended in media into the upper compartments, and pipette chemotactic factors and controls into the bottom compartments. The chemotactic factors can be used in various dilutions to get a dose-response curve. The controls are generally of three kinds: (a) negative, when the same media that is used to suspend the cells is also used below the membrane, (b) chemokinetic, when a chemotactic factor is placed at the same concentration in the media with the cells and in the well on the opposite side of the membrane, and (c) positive, when a known chemoattractant is placed in the bottom wells. Chemokinetic controls allow the user to distinguish heightened random activity of the cells, due to contact with the chemotactic factor, from directional response in a concentration gradient of that chemotactic factor.
Cell activity assay apparatus can also be used to measure the response of cells of different origins-e.g., immune cells obtained from patients suffering from diseasesxe2x80x94to a chemotactic factor of known chemotactic activity. In this case the cells in question are interrogated by both a negative control and a known chemotactic factor to see if the differential response is depressed or normal.
Chemotactic activity is measured by establishing a stable concentration gradient in the cell activity assay apparatus; incubating it for a predetermined time; and then counting the cells that have migrated through the membrane (or into the membrane). A comparison is then made between the activity of the cells in a concentration gradient of the chemotactic factor being tested, and the activity of the cells in the absence of the concentration gradient.
In one type of cell activity assay apparatus and method, the chemotactic response is measured by physically counting the number of cells on the membrane surface closest to the chamber containing the chemical agent. An example of this type of cell activity assay apparatus is described in U.S. Pat. No. 5,210,021 (Goodwin, Jr.), which is hereby incorporated by reference. One prior art method of obtaining quantitative data is to remove the membrane from the cell activity assay apparatus, remove the cells from the membrane surface closest to the chamber containing the original cell suspension, fix and stain the remaining cells, and then observe and count the stained cells under a microscope. Because of the time and expense associated with examining the entire membrane, only representative areas of the membrane are counted, rendering results less accurate than would otherwise be the case if the entire membrane were examined and counted.
Cell activity assays using a disposable ninety-six well microplate format, for example the ChemoTx(trademark) System (available from Neuro Probe, Inc., Gaithersburg, Md.), is amenable to different methods of quantification of results. The manual staining and counting method described above can be used, but is not recommended due to the time involved. A preferred method is to centrifuge the microplate with filter attached, such that, the cells that have migrated through the filter are deposited onto the bottom of the lower wells. The cells are then stained with MTT, MTS (available from Promega, Madison, Wis.), or similar dye, and then read in a standard automated laboratory densitometric reader (sometimes referred to as an Elisa plate reader).
Another method of obtaining quantitative data with this apparatus is to dye the cells with a fluorescent material, e.g., Calcein AM (available from Molecular Probes, Eugene, Oreg.); centrifuge the migrated cells into the microplate; and count cells with an automatic fluorescence plate reader (eg., Cytofluor available from PE Biosystems, Foster City, Calif., Victor2 available from EGandG Wallac, Gaithersburg, Md., or fmax available from Molecular Devices, Sunnyvale, Calif.). The automatic plate reader excites the fluorescent dye in the migrated cells with one wavelength of light and reads the light emitted at a second wavelength. Alternatively, the cells that have not migrated are removed from the top of each site, and the plate with framed membrane attached is read in the automatic fluorescent plate reader without spinning the cells into the plate, thereby counting the cells that have fallen off the filter into the lower well as well as those on the bottom of the membrane and in the pores of the membrane.
In another type of chemotaxis apparatus, illustrated by U.S. Pat. No. 5,601,997 (Tchao), the chemotactic response is also measured by labeling the cells with a fluorescent dye, as above. However, in Tchao the membrane is made of film opaque to the excitation and emission wavelengths of the fluorescent dye so that the cells on one side of the membrane can be counted without removing the cells from the opposite side. Tchao""s method is an example of a kinetic assay. In such assays, the side of the membrane toward which the cells are migrating is illuminated with the excitation wavelength of the dye, and the cells on that side are periodically counted by measuring the intensity of light emitted in the emission wavelength. This gives the researcher data on the rate at which cells are moving through the membrane. The membrane must be opaque because the researcher cannot remove the cells from the side from which they originated without ending the assay, which makes a kinetic study impossible.
Definitions and Abreviations
Abbreviations
1. xe2x80x9cElectromagnetic radiationxe2x80x9d is herein abbreviated to xe2x80x9cER.xe2x80x9d
2. xe2x80x9cPore diameterxe2x80x9d is herein abbreviated to xe2x80x9cpd.xe2x80x9d
3. xe2x80x9cMembrane thicknessxe2x80x9d is herein abbreviated to xe2x80x9cmt.xe2x80x9d
4. xe2x80x9cRadius of curvaturexe2x80x9d is herein abbreviated to xe2x80x9crc.xe2x80x9d
5. xe2x80x9cHigh throughput screeningxe2x80x9d is herein abbreviated to xe2x80x9cHTS.xe2x80x9d
6. xe2x80x9cCell-based high throughput screeningxe2x80x9d is herein abbreviated to xe2x80x9cCBHTS.xe2x80x9d
7. xe2x80x9cNanometerxe2x80x9d is herein abbreviated to xe2x80x9cnm.xe2x80x9d
8. xe2x80x9cmicrolitersxe2x80x9d is herein abbreviated to xe2x80x9cxcexcl.xe2x80x9d
9. xe2x80x9cmicrogramsxe2x80x9d is herein abbreviated to xe2x80x9cxcexcg.xe2x80x9d
10. xe2x80x9cCoefficient of variationxe2x80x9d is herein abbreviated to xe2x80x9cCV.xe2x80x9d
11. xe2x80x9cCell activity assay apparatusxe2x80x9d is herein abbreviated to xe2x80x9cCAAAxe2x80x9d
12. xe2x80x9ctccxe2x80x9d herein abbreviates xe2x80x9ctotal count of cellsxe2x80x9d introduced at a site.
13. xe2x80x9cU1xe2x80x9d herein abbreviates the quantity of light emitted from the upper volume of a site of a CAAA at the start of an assay. This is proportional to tcc.
14. xe2x80x9cL1xe2x80x9d herein abbreviates the quantity of light emitted from the lower volume of a site of a CAAA at the start of an assay, known as the background.
15. xe2x80x9cU2xe2x80x9d herein abbreviates the quantity of light emitted from the upper volume of a site of a CAAA at the end of an assay.
16. xe2x80x9cL2xe2x80x9d herein abbreviates the quantity of light emitted from the lower volume of a site of a CAAA at the end of an assay.
17. xe2x80x9cCL2xe2x80x9d herein abbreviates the quantity of light emitted from the lower volume of a site of a CAAA, L2, by subtracting the background L1.
18. xe2x80x9ccmcxe2x80x9d herein abbreviates xe2x80x9ccompletely migrated cellsxe2x80x9d which is proportional to CL2.
19. xe2x80x9ccmc%xe2x80x9d herein abbreviates percent of cmc with respect to the total cells introduced at that site, that is cmc/tcc.
20. xe2x80x9cpmcxe2x80x9d herein abbreviates xe2x80x9cpartially migrated cellsxe2x80x9d which is proportional to U1-(CL2+U2).
21. xe2x80x9cpmc%xe2x80x9d herein abbreviates percent of cells that partially migrated with respect to the total cells introduced at that site, that is pmc/tcc.
22. xe2x80x9cmcxe2x80x9d herein abbreviates xe2x80x9cmigrated cellsxe2x80x9d which equals cmc+pmc.
23. xe2x80x9cmc%xe2x80x9d herein abbreviates percent of mc, that is mc/tcc.
Definitions
1. xe2x80x9cchamber,xe2x80x9d xe2x80x9cwellxe2x80x9d or xe2x80x9cvolume,xe2x80x9d as used herein with respect to CAAA means the three-dimensional area of the CAAA for holding fluid samples.
2. xe2x80x9coff-axis,xe2x80x9d as used herein with respect to pores in membranes, means pores that are incident to the membranes, when held flat, with an angle of incidence greater than 0xc2x0 and less than 90xc2x0.
3. xe2x80x9cStrictly normal,xe2x80x9d as used herein with respect to a beam of ER and a flat surface, means the entire beam of ER is perpendicular to that surface.
4. xe2x80x9cxcex2-normal,xe2x80x9d as used herein with respect to ER and a flat surface, means a beam of ER some of which is strictly normal to that surface, and the remainder of which is incident within a range of angles where xcex2 is the largest such angle and xcex2 less than 90 degrees. In other words, a xcex2-normal beam of ER with respect to a surface is a cone of light having xcex2 as the largest angle of incidence as shown in FIG. 2.
5. xe2x80x9cSubstantially normal,xe2x80x9d as used herein with respect to ER and a flat surface, means that the ER is xcex2-normal to said surface and xcex2 is less than 15xc2x0. (This is in recognition that most detection and quantification systems employed in cell activity assays have detection beams where xcex2 is less than 15xc2x0.)
6. xe2x80x9cDetection beam,xe2x80x9d as used herein with respect to ER, means the ER of an automated reader or detection and quantification system directed at the sites of the apparatus within a specified cone, from a specified distance and from a specified aperture.
7. xe2x80x9cxcex2-normal detection beam,xe2x80x9d as used herein, means a detection beam using xcex2-normal ER.
8. xe2x80x9cIdeal opaque,xe2x80x9d as used herein with respect to a film, means film that stops or blocks all ER.
9. xe2x80x9cR-opaque,xe2x80x9d as used herein with respect to film or membrane, means a film or membrane that stops ER in the wavelength range R, where R is specified by a pair of numbers in brackets which delimits a range of ER wavelengths expressed in nanometers.
10. xe2x80x9cR-opaque @P%,xe2x80x9d as used herein with respect to film or membrane, means film or membrane that stops greater than P% of ER in range R, where P is a decimal number between 0 and 100 and represents the percent of ER that is blocked. For example, one film used in embodiments of this invention blocks more than 99.0% of ER in the wavelength range between 400 and 580 nanometers. The same film blocks 99.9% in the ER ranges between 480 and 490, and between 510 and 540 nanometers. This film is herein referred to as xe2x80x9c[400-580]-opaque @99.0%xe2x80x9d and xe2x80x9c[480-490]-opaque @99.9%xe2x80x9d and xe2x80x9c[510-540]-opaque @99.9%.xe2x80x9d
11. xe2x80x9cIdeal-opaque,xe2x80x9d as used herein with respect to membranes, means membranes such that (a) the film from which such membranes are made is ideal opaque, (b) the pores of such membranes are straight and parallel, and (c) said pores are positioned or angled such that when the membranes are flat, strictly normal ER cannot pass straight through the pores.
12. xe2x80x9cxcex2-normal-opaque,xe2x80x9d as used herein with respect to a membranes, means membranes such that no xcex2-normal ER can pass straight through any pore.
13. xe2x80x9cSubstantially opaque,xe2x80x9d as used herein with respect to membranes, means membranes such that no substantially normal ER can pass straight through their pores.
14. xe2x80x9cGeometrically R-opaque @P%,xe2x80x9d as used herein with respect to membranes, means membranes that are (a) made of film that is R-opaque @P%, and (b) no strictly normal ER passes straight through any pore of the membrane. As shown in FIG. 1, the least angle of incidence xcex1 of the off-axis pores of such a membrane must satisfy the following equation:
xcex1 greater than xcfx86 where xcfx86=sinxe2x88x921(pd/mt).
15. xe2x80x9cxcex2-normal R-opaque @P%,xe2x80x9d as used herein with respect to membranes, means (a) the film of the membrane is R-opaque @P%, and (b), no xcex2-normal ER passes straight through any pore. As shown in FIG. 2, the least angle of incidence xcex1 of the off-axis pores of such a membrane must satisfy the following equation:
xcex1 greater than (xcex2+xcex8), where xcex8=sinxe2x88x921([pdxc3x97cos xcex2]/mt).xe2x80x83xe2x80x83(2)
16. xe2x80x9cSubstantially R-opaque @P%,xe2x80x9d as used herein with respect to membranes, means membranes that are (a) made of R-opaque film @P%, and (b) allow no substantially normal ER to pass straight through any pore. Thus the least angle of incidence xcex1 of the off-axis pores of such a membrane must satisfy equation (2), above, for xcex2 less than 15xc2x0.
17. xe2x80x9cSubstantially perpendicular,xe2x80x9d as used herein with respect to pores in membranes, means pores that are incident to the membranes, when held flat, with an angle of incidence less than 15xc2x0.
Note that membranes that are R-opaque @P% are not necessarily substantially R-opaque @P%, xcex2-normal R-opaque @P%, or geometrically R-opaque @P%, since they may have perpendicular pores which allow substantially normal light to pass straight through them. Note that a membrane suitable for cell activity assays will preferably have P greater than 99.0%, and the pore diameter will be greater than 3 microns so that the open area of the membrane formed by the pores will be larger than 2% of the membrane. More specifically, membranes for these applications cannot be R-opaque at 99% if the pores are substantially perpendicular because ER substantially perpendicular to the surface will pass straight through the pores. If, on the other hand, the pores are sufficiently off-axis, and all other aspects remain the same, the membrane can
The present invention provides CAAA employing membranes and methods for using the CAAA for HTS, CBHTS, and cell based screening, as well as basic research in cell activity. In particular the present invention is a CAAA using a class of membranes that are substantially R-opaque @P%. The present invention also includes the membranes used in the CAAA and methods for their fabrication.
Because the membranes of this invention are substantially R-opaque @P% to the ER wavelengths of the instruments used for detection and quantification with CAAA, detection and counting of cells from one side of the membrane will not be influenced by cells on the opposite side of the membrane or by simultaneous or by subsequent detection and counting of cells on the other side of the membrane. This yields more accurate results than can be obtained with prior art methods. In particular, this allows the use of a method of detection and quantification that eliminates the errors due to both the volumetric inaccuracies of pipetting and the variations in the distribution of cells in the media in which the cells are suspended. This lowers the CV of the assays such that they are appropriate for HTS and CBHTS in drug discovery and development.
Tchao""s membrane is made of film opaque to the wavelengths of excitation and emission of some fluorescent dyes. That is, the Tchao membrane is R-opaque @P%, where R is the range of wavelengths used by the detection and quantification system, and P is the percent of light blocked. The Tchao membrane, however, is specifically required to have substantially perpendicular transverse pores. It is therefore neither a xe2x80x9cgeometrically R-opaque @P%xe2x80x9d membrane, nor a xe2x80x9cxcex2-normal R-opaque @P%xe2x80x9d membrane, where xe2x80x9cxcex2xe2x80x9d is the largest angle of incidence of light in the detection beam, and xe2x80x9cRxe2x80x9d is a range of wavelengths of light. It is therefore not a xe2x80x9csubstantially R-opaque @P%xe2x80x9d membrane. ER from detection beams of standard detectors will pass straight through the pores since they are substantially perpendicular. Therefore, with membranes commonly used for cell activity assays which have between 5% and 15% open area (the total area of the pores), the amount of light passing through the pores of the membrane is significant. The transmission of wavelengths normal to the surface of the membrane from the excitation beam will pass through substantially perpendicular pores, and cells on the opposite side of the membrane from the detection beam will be counted if they are over a pore. In other words, light that is xcex2-normal where xcex2 is less than 15xc2x0 will cross the membrane, excite cells that are over pores which will emit light and be counted (since that light will pass back through the substantially perpendicular pores). Thus cells that have not migrated through the membrane will be stimulated to emit ER and will be counted, reducing the accuracy of the results. For kinetic assays, this is not a problem for two reasons. First, the membrane""s open area is only between 5% and 15%, and the number of cells that are used for an assay can be set so that they cover only 10% of the membrane. This reduces the number of a cells starting out over pores. Secondly, in a kinetic study, the important parameter is the rate of change, and the fact that cells on the origination side of the membrane are counted when they are over pores means only that the detector will count them much earlier than it would with an opaque membrane made out of the same film with pores that are off axis so that the excitation beam cannot pass directly through them. On the other hand, for assays that measure more than just kinetics or do not measure kinetics, and where the method involves counting all the cells on both sides of the membrane at different points in the assay, as in the methods described below, having substantially perpendicular pores will decrease the accuracy of the assay, and increase the CV significantly.